Prevention of the β-amyloid peptide-induced inflammatory process by inhibition of double-stranded RNA-dependent protein kinase in primary murine mixed co-cultures.

Inflammation may be involved in the pathogenesis of alzheimer's disease (ad). there has been little success with anti-inflammatory drugs in ad,while the promise of anti-inflammatory treatment is more evident in experimental models. a new anti-inflammatory strategy requires a better understanding of molecular mechanisms. among the plethora of signaling pathways activated by β-amyloid (aβ) peptides,the nuclear factor-kappa b (nf-κb) pathway could be an interesting target. in virus-infected cells,double-stranded rna-dependent protein kinase (pkr) controls the nf-κb signaling pathway. it is well-known that pkr is activated in ad. this led us to study the effect of a specific inhibitor of pkr on the aβ42-induced inflammatory response in primary mixed murine co-cultures,allowing interactions between neurons,astrocytes and microglia. primary mixed murine co-cultures were prepared in three steps: a primary culture of astrocytes and microglia for 14 days,then a primary culture of neurons and astrocytes which were cultured with microglia purified from the first culture. before exposure to aβ neurotoxicity (72 h),co-cultures were treated with compound c16,a specific inhibitor of pkr. levels of tumor necrosis factor-α (tnfα),interleukin (il)-1β,and il-6 were assessed by elisa. levels of pt451-pkr and activation of iκb,nf-κb and caspase-3 were assessed by western blotting. apoptosis was also followed using annexin v-fitc immunostaining kit. subcellular distribution of pt451-pkr was assessed by confocal immunofluorescence and morphological structure of cells by scanning electron microscopy. data were analysed using one-way anova followed by a newman-keuls' post hoc test in these co-cultures,pkr inhibition prevented aβ42-induced activation of iκb and nf-κb,strongly decreased production and release of tumor necrosis factor (tnfα) and interleukin (il)-1β,and limited apoptosis. in spite of the complexity of the innate immune response,pkr inhibition could be an interesting anti-inflammatory strategy in ad.